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Human Dermal Fibroblasts in Response to the Three
Main Species of Borrelia burgdorferi sensu lato
Mariam Meddeb, Wassila Carpentier, Nicolas Cagnard, Sophie Nadaud,
Antoine Grillon, Cathy Barthel, Sylvie de Martino, Benoît Jaulhac, Nathalie
Boulanger, Frédéric Schramm
To cite this version:
Mariam Meddeb, Wassila Carpentier, Nicolas Cagnard, Sophie Nadaud, Antoine Grillon, et al.. Ho-mogeneous Inflammatory Gene Profiles Induced in Human Dermal Fibroblasts in Response to the Three Main Species of Borrelia burgdorferi sensu lato. PLoS ONE, Public Library of Science, 2016, 11 (10), pp.e0164117. �10.1371/journal.pone.0164117.s004�. �hal-01380244�
Homogeneous Inflammatory Gene Profiles
Induced in Human Dermal Fibroblasts in
Response to the Three Main Species of
Borrelia burgdorferi sensu lato
Mariam Meddeb1, Wassila Carpentier2, Nicolas Cagnard3, Sophie Nadaud4, Antoine Grillon1, Cathy Barthel1, Sylvie Josiane De Martino1,5, Benoıˆt Jaulhac1,5,
Nathalie Boulanger1,5, Fre´de´ric Schramm1¤*
1 EA7290 Early Bacterial Virulence: Lyme borreliosis Group, FMTS, Universite´ de Strasbourg, Strasbourg, France, 2 Plate-forme Post-Ge´nomique P3S, Universite´ Pierre et Marie Curie, Faculty of Medicine, Paris, France, 3 Plateforme Bio-informatique, Universite´ Paris Descartes, Structure Fe´de´rative de Recherche Necker, INSERM US24/CNRS UMS 3633, Paris, France, 4 INSERM UMR 1166, Universite´ Pierre et Marie Curie, Universite´ Paris 06, Paris, France, 5 French National Reference Center for Borrelia, University Hospital, Strasbourg, France
¤ Current address: Institut de Bacte´riologie, EA 7490, Faculte´ de Me´decine, Universite´ de Strasbourg, Strasbourg, France
*fredericschramm@unistra.fr
Abstract
In Lyme borreliosis, the skin is the key site for bacterial inoculation by the infected tick and for cutaneous manifestations. We previously showed that different strains of Borrelia
burg-dorferi sensu stricto isolated from tick and from different clinical stages of the Lyme
borrelio-sis (erythema migrans, and acrodermatitis chronica atrophicans) elicited a very similar transcriptional response in normal human dermal fibroblasts. In this study, using whole transcriptome microarray chips, we aimed to compare the transcriptional response of nor-mal human dernor-mal fibroblasts stimulated by 3 Borrelia burgdorferi sensu lato strains belonging to 3 main pathogenic species (B. afzelii, B. garinii and B. burgdorferi sensu stricto) in order to determine whether “species-related” inflammatory pathways could be identified. The three Borrelia strains tested exhibited similar transcriptional profiles, and no species-specific fingerprint of transcriptional changes in fibroblasts was observed. Con-versely, a common core of chemokines/cytokines (CCL2, CXCL1, CXCL2, CXCL6,
CXCL10, IL-6, IL-8) and interferon-related genes was stimulated by all the 3 strains. Dermal fibroblasts appear to play a key role in the cutaneous infection with Borrelia, inducing a homogeneous inflammatory response, whichever Borrelia species was involved.
a11111
OPEN ACCESS
Citation: Meddeb M, Carpentier W, Cagnard N,
Nadaud S, Grillon A, Barthel C, et al. (2016) Homogeneous Inflammatory Gene Profiles Induced in Human Dermal Fibroblasts in Response to the Three Main Species of Borrelia burgdorferi sensu lato. PLoS ONE 11(10): e0164117. doi:10.1371/journal.pone.0164117
Editor: Brian Stevenson, University of Kentucky
College of Medicine, UNITED STATES
Received: May 31, 2016 Accepted: September 19, 2016 Published: October 5, 2016
Copyright:© 2016 Meddeb et al. This is an open access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: The author(s) received no specific
funding for this work.
Competing Interests: The authors have declared
that no competing interests exist.
Abbreviations: IFN, interferon; EM, erythema
migrans; ACA, acrodermatitis chronica
Introduction
Lyme borreliosis is caused by spirochetes of the Borrelia burgdorferi sensu lato (sl) group that are transmitted by Ixodes spp. ticks: the bacteria are inoculated intradermally, multiply locally and eventually spread to various secondary organs throughout the body, especially the skin, the nervous system and the joints, inducing inflammatory lesions [1]. Lyme borreliosis is the most common tick-borne infectious disease in North America [2] and in countries with mod-erate climates in Eurasia [3]. Whereas B. burgdorferi sensu stricto (ss) is the only main Borrelia species responsible for Lyme borreliosis manifestations in North America, at least 8 species can be pathogenic for humans in Europe, with 5 clearly pathogenic species—B. burgdorferi ss, B.
afzelii, B. garinii, B. bavariensis [4] and B. spielmanii [5]—, and 3 rarely, if at all, pathogenic species for humans—B. bissettii [6,7], B. lusitaniae [8] and B. valaisiana [9]. The erythema migrans (EM), first clinical manifestation of Lyme borreliosis and considered the hallmark of the disease, can be caused by any pathogenic Borrelia species of the B. burgdorferi sl group. In contrast, acrodermatitis chronica atrophicans (ACA) is typically associated with B. afzelii, neu-rological manifestations most commonly involve B. garinii and arthritis is preferentially related to B. burgdorferi ss [10].
The skin is a key interface in Lyme borreliosis since it represents both the inoculation site of the spirochetes, a potential filter to select specific invasive clones injected at the tick-bite site [11,12], and also the target of clinical manifestations at various stages of the disease [13]. How-ever, factors determining the limitation of the disease to a benign localised EM or the appear-ance of systemic manifestations and the way the bacteria escape the local cutaneous immunity, spread, and reach distal target organs are unknown. Thus, understanding the interaction of the bacteria with skin cells is essential. The role of the skin’s immune cells in anti-Borrelia defense mechanisms was addressed in several previous works [14–16], but the skin’s resident cells (ker-atinocytes and fibroblasts) were also shown to be sensors of danger [13] and to play a key role in the anti-Borrelia response [17,18]. In particular, we previously showed that Borrelia induces the transcription of numerous inflammatory genes in normal human dermal primary fibro-blasts (NHDF) [19], including chemokines and cytokines and many transcription factors and components of the extracellular matrix. As the 3 B. burgdorferi ss strains tested in our model were isolated from different stages of the human infection and elicited very similar transcrip-tional modulation profiles, we concluded that Borrelia pathotype has little influence on the fibroblasts’ response. Since the 3 strains used in our previous study belonged to the same spe-cies, in this new study, we wanted to assess whether the species of Borrelia could influence the transcriptional modulations of fibroblasts. For this purpose, we tested 3 Borrelia strains, all iso-lated from erythema migrans lesions and belonging to 3 different pathogenic species (B. afzelii,
B. garinii and B. burgdorferi ss) in our NHDF in vitro model, and compared the transcriptional
profiles induced by the different species using whole transcriptome microarrays.
Materials and Methods
Spirochetes strains
Three different strains of Borrelia representing the 3 major European species were chosen among the collection of clinical isolates maintained in the Institute of Bacteriology of Stras-bourg: B. afzelii strain IBS17, B. garinii strain IBS6 and B. burgdorferi ss strain IBS19. These 3 strains were isolated from patients presenting a solitary EM as a unique clinical manifestation (Table 1). Each strain was used at passage 7, cultured in BSK-H medium (Sigma, Saint Quentin Fallavier, France) at 33°C with 5% CO2, and washed twice before the assays as
previ-ously described [19]. Careful washing of Borrelia prior to every coincubation experiment was a
fibroblasts; MOI, multiplicity of infection; SDs, standard deviations; QRT-PCR, semiquantitative reverse transcription PCR.
prerequisite before every stimulation experiment since, in our preliminary experiments, we observed that the BSK medium by itself induces a nonspecific transcriptional response in human dermal fibroblasts that was significantly higher than that of negative control fibroblasts (data not shown).
Fibroblast culture and stimulation
Primary human dermal fibroblasts (NHDF, Promocell, Heidelberg, Germany) were main-tained in FGM2 medium (detailed characteristics of the fibroblast’s batches are presented in
Table 2). To stimulate the cells, fibroblasts were used at passage 5 and seeded at 7.5 x 104per well in a 24-well plate. At confluence and one day before Borrelia activation, FGM2 medium was replaced by FGM medium without fetal calf serum. If not otherwise stated, fibroblasts were stimulated with B. burgdorferi spirochetes at a multiplicity of infection (MOI) of 100:1 (100 Borrelia per fibroblast) for 24 hours.
In order to provide evidence that our experimental methods and specifically the coincuba-tion of Borrelia and fibroblasts in a BSK-free medium was compatible with the viability of
Bor-relia, viability experiments were conducted: Borrelia were washed twice, suspended in FGM
medium and either incubated as is (at a concentration of 2.5 x 106Borrelia per ml), or
coincu-bated with fibroblasts at a MOI of 100:1. Twenty-four hours later, the culture medium (consist-ing of either Borrelia in FGM medium or Borrelia in FGM medium coincubated with human dermal fibroblasts), was recollected and instilled in BSK medium. We observed that both con-ditions provided viable Borrelia 96 hours later (data not shown).
IL-8 ELISA
IL-8 secretion levels were measured in culture supernatants of unstimulated and Borrelia-stimu-lated cells by ELISA. The protocol was based on sandwich techniques, as described by the manu-facturer (R&D systems, Lille, France). Experiments of cell stimulation with spirochetes were carried out twice in independent experiments. Results are presented as means ± standard devia-tions (SDs) of triplicate values and are representative of the two independent experiments.
Table 1. Origin of the Borrelia strains used in this study.
n˚ Species Geographical origin, Country Anatomical origin Associated symptoms
IBS6 B. garinii Strasbourg, France Skin biopsy of EMa none
IBS17 B. afzelii La Roche Guyon, France Skin biopsy of EM none
IBS19 B. burgdorferi ss Metz, France Skin biopsy of EM none
a
Erythema migrans.
doi:10.1371/journal.pone.0164117.t001
Table 2. Characteristics of the fibroblast batches used in this study.
NHDF Batch n˚ Anatomical origin Agea Sexe Race
#1 3021904 breast 20 female caucasian
#2 3022702 eyelid 68 female caucasian
#3 10118012 temple 70 male caucasian
#4 10215061 cheek 56 female caucasian
#5 20612062 cheek 60 male caucasian
#6 70711061 lip 32 female caucasian
a
Age is given in years.
RNA extraction and semiquantitative real time RT-PCR
After removal of the supernatant, fibroblasts were directly resuspended in Trizol (Invitrogen, Cergy-Pontoise, France) and stored at -80°C until use. RNA extraction was performed with RN easy mini-kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s protocol, includ-ing treatment with DNase (Ambion, Courtaboeuf, France). Two μg of total RNA were reverse-transcribed with the Superscript II first-strand synthesis system (Invitrogen, Cergy-Pontoise, France). Semiquantitative reverse transcription PCR (QRT-PCR) was done on an ABI Prism 7000 (Applied Biosystems, Courtaboeuf, France) with specific primers (S1 Table). Expression levels of all transcripts studied were normalised to housekeeping gene level and the relative changes in gene expression were compared with those of untreated cells using the 2-ΔΔCt method. Two housekeeping genes were tested: β-actin and the RNA polymerase II genes [20].
Microarray analysis
Sample generation and DNA microarray hybridisation and analysis. The design of the
microarray experiment included 4 different analytical conditions: unstimulated fibroblasts, and fibroblasts stimulated with B. burgdorferi ss IBS19, B. garinii IBS6 and B. afzelii IBS17 strains at MOI 100:1 for 24h. For each analytical condition, a total of 6 biological replicates rep-resented by 6 different batches of human dermal primary fibroblasts (Table 2) were used. A total of 24 biological samples was therefore processed for this microarray experiment.
The HumanHT-12 V4.0 expression BeadChip (Illumina Inc., Cambridge, UK), comprising 47,232 transcripts was used to generate gene expression profiles of unstimulated and Borrelia-stimulated fibroblasts. RNA was extracted using RNeasy Mini kit (Qiagen, Courtaboeuf, France). Each sample contained 50–200 ng RNA, and the quality was assessed with the 2100 Agilent Bioanalyzer. The cRNA was synthesized, amplified and purified using the Illumina TotalPrep RNA Amplification Kit (Ambion, Foster City, CA, USA) following manufacturer recommendations. Briefly, 200 ng of RNA was reverse transcribed. After second strand synthe-sis, the cDNA was transcribed in vitro and cRNA labelled with biotin-16-UTP. Labelled probe hybridisation to the Beadchips was carried out using Illumina’s protocol. The Beadchips were scanned on the Illumina IScan using Illumina IScan image data acquisition software. Illumina GenomeStudio software was used for preliminary data analysis, data normalisation and quality controls. Raw microarray intensity data was background subtracted and normalised using the “normalise quantiles” function. We used the detection P-values as flags, flag = 0, if P> 0.05 and flag = 1, if P 0.05. Each tested probe list was created after filtering probes flagged ‘1’ for at least half of the chips involved in the comparison. The group comparisons were made using Student’s t-test. We filtered the resulting P-values at 5% and at a 2-fold threshold for functions and pathway analyses. For each particular sample, a given transcript was considered regulated if the mean fold change exceeded 1.2 in comparison with the “unstimulated” condition. The microarray data have been deposited in the GEO repository (http://www.ncbi.nlm.nih.gov/ geo/) with the record number GSE77058. Microarray experiments were performed according to the MIAME guidelines [21].
Results
Human dermal fibroblasts stimulated by B. burgdorferi ss IBS19, B.
garinii IBS6 and B. afzelii IBS17 secrete IL-8
When coincubated with human primary dermal fibroblasts, different B. burgdorferi ss strains were shown to induce a pro-inflammatory response with IL-8 synthesis [18,19]. To analyze the impact of the Borrelia species in our experimental model, we first measured the IL-8
synthesis by fibroblasts stimulated by B. burgdorferi ss IBS19, B. garinii IBS6 and B. afzelii IBS17. The 6 batches of fibroblasts tested in our study were able to produce the IL-8 chemo-kine. Even though absolute levels of IL-8 were highly variable between batches of cells tested, the levels of IL-8 synthesis obtained with the 3 Borrelia strains used for stimulation were com-parable in each batch of fibroblasts (Fig 1).
A complete kinetic and titration study was performed for fibroblasts batch #1, and showed that the chemokine was secreted in a dose- and time-dependent manner for each of the 3 tested
Borrelia strains (Fig 2). Similar profiles for titration experiments were obtained with fibroblasts expressing lower levels of IL-8 (S1 Fig).
Global fibroblast transcriptional responses to B. burgdorferi ss IBS19, B.
garinii IBS6 and B. afzelii IBS17
Transcriptional modifications were studied by the Human HT12 BeadChip microarray, allow-ing whole transcriptome analysis. Statistical analysis was then performed after normalization of the raw results obtained for the 6 tested biological replicates (fibroblasts batch #1 to fibro-blasts batch #6) and the 4 analytical conditions (unstimulated, B. burgdorferi ss IBS19-stimu-lated, B. garinii IBS6-stimulated and B. afzelii IBS17-stimulated) by comparing Borrelia-stimulated vs unBorrelia-stimulated samples. Out of 47,231 transcripts present on the chip, 1,968 tran-scripts (4.2%) were significantly regulated (1.2 fold vs unstimulated fibroblasts) by at least one the 3 tested strains. Among them, 1,291 transcripts were differentially regulated by B.
burg-dorferi ss IBS19 (675 up- and 616 down-regulated), 1,043 by B. garinii IBS6 (612 up- and 431
down-regulated), and 1,020 by B. afzelii IBS17 (494 up- and 526 down-regulated). The majority of these transcripts were regulated with a fold-change ranging from 1.2 to 2 for all 3 tested strains (Fig 3).
The global Borrelia-induced transcriptional modulation in fibroblasts was relatively similar in the 3 tested strains (similar number of up- and down-regulated genes) (Fig 3). Moreover, analyses of regulated genes showed that almost all the genes with high transcription modula-tion (2 fold vs unstimulated fibroblasts) were consistently stimulated by the 3 tested species (Fig 4B). The genes induced by only one of the tested species displayed mild transciptional modulations (between 1.2 and 2 fold vs unstimulated fibroblasts). However, it is noteworthy that the global level of modification induced by B. garinii IBS6 appeared significantly higher
Fig 1. Measurement of IL-8 secretion by 6 different batches of fibroblasts coincubated with strains of 3 different species of the B. burgdorferi sl group. IL-8 secretion of fibroblasts stimulated by B. burgdorferi ss (Bb ss) IBS19, B. garinii IBS6, and B. afzelii IBS17 at MOI of 100:1 after 24h of stimulation. Each bar shows the mean±SDs of triplicate values (technical replicates) obtained for each batch of fibroblasts— fibroblasts batch #1 to fibroblasts batch #6 (biological replicates). The results are representative of 2 independent experiments.
than that of B. afzelii IBS17 (P = 0.0007) and B. burgdorferi ss IBS19 (P = 0.0001), while the amplitude of activation induced by B. afzelii IBS17 and B. burgdorferi ss IBS19 did not statisti-cally differ (Fig 4A).
Highly up-regulated transcriptional responses are largely representative
of pro-inflammatory pathways
We did not identify relevant specific strain-related transcriptional pathways. In contrast, we observed that the 3 strains belonging to 3 different species of the B. burgdorferi sl group elicited very similar transcriptional profiles in primary human dermal fibroblasts (see the global func-tional analyses and the signalisation pathway analyses results inS2andS3Figs). We then focused on co-regulated genes presenting the highest intensity of up-regulation. Among them, a common core of 42 genes were highly up-regulated in response to stimulation by all 3
Borre-lia species. These mainly included pro-inflammatory genes involved in the innate immune
response (Table 3). High levels of chemokines/cytokines were induced (CCL2, CXCL1, CXCL2, CXCL6, CXCL10, IL-6, IL-8), with fold-change values up to 20 for IL-8 in fibroblasts stimulated by B. garinii IBS6, along with a large number of genes representative of intracellular signalling pathways that sustain inflammatory responses, such as NF-κB- and interferon (IFN)-related genes. Whichever strain we tested, the IFN signalling and the activation of IRF by cytosolic pattern recognition receptors were found to be the two most overexpressed path-ways in the signalisation pathway analyses (S2 Fig). Several other genes related to the
Fig 2. Kinetics and titration of IL-8 secretion by fibroblasts co-incubated with different species of the B. burgdorferi sl group. (A-C) Kinetic studies of 8 secretion of fibroblasts stimulated by B. burgdorferi ss (Bb ss) IBS19, B. garinii IBS6, and B. afzelii IBS17 at MOI of 100:1. (D-F) Levels of IL-8 secretion by fibroblasts stimulated by increasing concentrations (MOI of 1:1 = 1B, MOI of 10:1 = 10B, 50:1 = 50B, and 100:1 = 100B) of the 3 Borrelia strains at 24 hours. NEG: unstimulated fibroblasts. (A-F) Each bar shows the mean±SDs of duplicate values obtained for fibroblasts batch #1. doi:10.1371/journal.pone.0164117.g002
metabolism, such as SOD2, were up-regulated by all 3 Borrelia species. Among all these genes, 7 of them (CXCL1, IL-6, IL-8, OAS2, STAT1, IFIH1 and SOD2) were already identified in our previous study as belonging to a common core of up-regulated genes stimulated in fibroblasts by different strains of the same species B. burgdorferi ss (strain N40 isolated from tick, strains Pbre and 1408 isolated from patients affected by EM or ACA respectively). When focusing on the most highly down-regulated genes, a common core of 9 genes was found to be down-regu-lated in response to all 3 Borrelia species. These genes are mainly involved in the cell division process (Table 4).
A focus on selected genes among those found to be differentially
regulated by microarray analyses
The mRNA transcription of selected genes found to be up-regulated by microarrays (IL-8, CXCL1, IL-6 and SOD2) was analyzed 24h after Borrelia stimulation at MOI 100:1. Using QRT-PCR assays, we confirmed the strong up-regulation of the genes encoding IL-8, CXCL1, IL-6, and SOD2 in all 6 batches of fibroblasts and the three Borrelia strains. The QRT-PCR results normalised to the β-actin (Fig 5A) are confirmed by normalising them to the expression of the RNA polymerase II (Fig 5B), another housekeeping gene known to be stable under vari-ous stimulatory conditions [20].
The mRNA transcription of selected genes found to be down-regulated by microarrays (UBE2C, KIF20A, TOP2A, CEP55 and CDC20) was also analyzed under the same experimental
Fig 3. Gene expression profiles obtained from human fibroblasts stimulated with the three species of the B. burgdorferi sl group. Number of genes differentially expressed during fibroblast stimulation with
Borrelia. The bars reflect the number of up-regulated genes (+) and down-regulated genes (-) for each strain.
The light dotted areas correspond to gene expression changes of 1.2–2.0-fold, the gray hatched areas correspond to changes of 2.0–5.0-fold and black areas represent changes5.0-fold.
conditions. Using QRT-PCR assays, we confirmed a moderate down-regulation of the genes encoding UBE2C, KIF20A, TOP2A, CEP55 and CDC20 in all 6 batches of fibroblasts induced by the three Borrelia strains (Fig 6).
Discussion
Since the 3 strains used in our previous study belonged to the same species, in this new study, we wanted to assess whether the species of Borrelia could influence the transcriptional modula-tions of fibroblasts. For this purpose, we tested 3 Borrelia strains, all isolated from erythema migrans lesions and belonging to 3 different pathogenic species (B. afzelii, B. garinii and B.
burgdorferi ss) in our NHDF in vitro model, and compared the transcriptional profiles induced
by the different species using whole transcriptome microarrays. Exploring the interaction between Borrelia and fibroblasts, we extended a previous experimental work [19] to a selection of Borrelia strains belonging to the three main pathogenic species of Borrelia in the North hemisphere, and we increased the range of analyzed transcripts by using a whole genome microarray experiment.
The technical choices of the present study are important. First of all, Borrelia strains used were cautiously selected. We tested the most representative Borrelia species by selecting one strain of each of the main European Borrelia species: B. afzelii, B. garinii, and B. burgdorferi ss [3]. In order to avoid any bias that could be related to the specific environment from which the strains were isolated, we selected 3 clinical Borrelia strains that were all isolated from the same type of lesion (EM), the most frequent human clinical manifestation. Bias related to the disseminating potential of Borrelia strains was also limited by the selection of strains associated with similar associated clinical features (i.e. without any general symptom suggestive of dissemination associated with EM). All strains tested in our study were
Fig 4. Intensity of transcriptional modifications. The distribution of regulated genes according to the level of regulation is expressed as fold-change in comparison to unstimulated cells. (A) Comparison of transcription levels between Borrelia strains.***P<0.001, NS: not significant, tested by ANOVA using GraphPad Prism6 software (GraphPad, San Diego, CA). (B) Transcription levels of common (stimulated by the 3 strains) and specific (stimulated by only one of the strains) genes. doi:10.1371/journal.pone.0164117.g004
Table 3. Genes consistently stimulated by the 3 Borrelia strains with fold-change values2.
Annotation B. afzelii (IBS17) B. garinii (IBS6) B. burgdorferi ss (IBS19) Description/Function Fold change p-value Fold change p-value Fold change p-value
Inflammation and innate immune response Chemokines/cytokines
CCL2 5.8 6.20E-04 8.2 2.10E-05 5.2 2.90E-04 Chemoattractant for monocytes and basophils
CXCL1 8.7 3.80E-05 13.4 2.10E-05 7.6 1.90E-03 GROα: chemoattractant for neutrophils
CXCL2 2.8 8.70E-04 3.6 3.30E-04 3.3 1.70E-03 Hematoregulatory chemokine
CXCL6 6 5.10E-04 7.6 1.80E-04 4.7 3.20E-04 Chemoattractant for neutrophils, strong
antibacterial activity
CXCL10 6.5 3.90E-02 4.4 9.90E-03 2.5 5.30E-04 Chemoattractant for monocytes and
T-lymphocytes
IL6 3.3 2.30E-03 6.2 1.10E-05 2.6 3.90E-03 Cytokine of the acute phase response
IL8 14.9 4.30E-04 20.2 1.90E-05 11 1.00E-03 Chemoattractant for neutrophils
IFN-related pathway
DDX58 2.2 7.50E-03 2.5 1.60E-03 2.7 4.90E-02 Innate immune receptor, induction of type I IFN
and pro-inflammatory cytokines
GBP2 2.1 1.30E-03 2.4 3.80E-03 2.4 6.30E-03 IFN-induced guanylate-binding protein 2: antiviral
activity
HERC5 3.2 6.90E-03 3.8 6.00E-04 3.7 1.20E-02 Positive regulator of innate antiviral response in
cells induced by IFN
IFI6 2.3 9.10E-03 3.3 1.20E-03 2.6 1.60E-03 IFN-α-inducible protein 6
IFI27 2.7 1.10E-03 5.6 3.60E-05 3.4 3.60E-03 IFN-α-inducible protein 27
IFI44 2.6 2.50E-03 3.2 7.60E-05 2.7 7.40E-03 IFN-induced protein 44
IFI44L 4.3 2.60E-03 7.4 1.90E-04 5.3 4.40E-03 IFN-induced
IFIH1 2.9 2.40E-03 3.3 4.60E-04 3.3 7.60E-03 Induction of type I IFN and pro-inflammatory
cytokines
IFIT1 5.2 5.00E-03 5.6 7.00E-05 5.1 1.00E-03 IFN-induced
IFIT2 6.4 1.80E-02 4.8 7.40E-05 4.8 3.00E-03 IFN-induced, activation of IRF by cytosolic pattern
recognition receptors
IFIT3 4.4 4.80E-03 5 8.60E-05 4.6 4.80E-03 IFN-induced, inhibition of cell migration and
proliferation
IFITM1 2.8 6.70E-04 4.6 9.10E-05 3.5 2.40E-04 IFN-induced transmembrane protein 1
IRF7 2.2 3.10E-03 2.8 9.60E-04 2.8 8.10E-04 IFN regulatory factor 7: transcriptional regulator of
type I IFN-dependent immune responses
ISG15 4.4 1.60E-03 6.9 2.10E-04 5.2 5.40E-04 NK cell proliferation, chemoattractant for
neutrophils, induce IFN-γ
MX1 8.4 5.10E-04 12.8 7.50E-05 11.1 4.60E-05 IFN-induced GTP-binding protein
MX2 3.5 1.10E-02 4.9 2.50E-04 4.2 3.20E-03 Interferon-induced GTP-binding protein
OAS1 2.9 6.80E-03 3.7 2.00E-04 3.3 3.70E-03 Oligoadenylate synthetase-1: IFN-induced, innate
immune response to viral infection
OAS2 4.1 3.00E-03 6 2.00E-04 5.1 4.90E-04 Oligoadenylate synthetase-2: IFN-induced, innate
immune response to viral infection
OAS3 2.8 1.10E-03 4.3 1.20E-04 3.5 6.30E-03 Oligoadenylate synthetase-3: IFN-induced,
dsRNA-activated antiviral enzyme
RSAD2 4 7.70E-03 4.5 3.40E-04 4.5 2.50E-02 IFN-inducible iron-sulfur cluster-binding antiviral
protein
STAT1 2.5 4.60E-04 3.6 5.50E-05 2.9 8.10E-03 Signal transducer of activation-1, up-regulate
genes in response to IFN type I, II or III NF-kB pathway
NFKBIA 2.2 3.20E-03 2.6 6.30E-04 2.2 3.90E-03 Inhibits the activity of dimeric NF-kappa-B/REL
complexes
cultured in strictly identical conditions, and were all used at low passage (P7) in order to avoid the loss of plasmid-encoded virulence factors [22]. The MOI of 100:1 Borrelia used for fibroblast stimulations in microarray and QRT-PCR analyses was chosen to be representa-tive of the physiologic spirochete load present in the dermis at the inoculation site a few days
Table 3. (Continued)
Annotation B. afzelii (IBS17) B. garinii (IBS6) B. burgdorferi ss (IBS19) Description/Function Fold change p-value Fold change p-value Fold change p-value
NFKBIZ 2.9 6.60E-03 3.2 2.50E-03 2.8 1.40E-02 Induction of inflammatory genes activated through
TLR/IL-1 receptor signalling Complement pathway
CFB 2.7 5.00E-03 4.2 6.30E-04 2.8 2.90E-03 Factor B which is part of the alternate pathway of
the complement system Miscellaneous
C15orf48 2.2 3.90E-04 3.5 1.10E-04 2.5 2.60E-03 Normal mucosa of esophagus-specific gene 1
protein
ECGF1 2 8.20E-03 2.5 2.00E-03 2 2.00E-03 Thymidine phosphorylase: angiogenic activity
EPSTI1 4.4 7.30E-04 6.1 5.40E-05 5.4 8.70E-04 Epithelial-stromal interaction protein 1
HERC6 2.3 5.60E-03 3.3 3.50E-04 2.8 1.60E-02 Probable E3 ubiquitin-protein ligase HERC6
IER3 2.3 3.50E-03 2.5 3.20E-03 2.5 1.10E-02 Role in the ERK signalling pathway
KYNU 2.7 6.20E-03 3.9 3.00E-03 2 2.90E-03 Putative uncharacterized protein KYNU:
kynureninase activity
PRIC285 2 4.90E-03 2.7 3.10E-03 2.7 4.60E-03 Helicase with zinc finger domain 2: acts as a
transcriptional coactivator for nuclear receptors
SLC39A8 3.3 1.20E-03 4.4 5.70E-04 2.8 3.10E-03 Zinc transporter ZIP8
SOD2 5 8.80E-05 6.7 3.30E-05 4.7 1.60E-04 Superoxide dismutase
TNFAIP6 5.3 3.40E-04 7.8 1.50E-05 4.9 1.20E-03 Cell-cell and cell-matrix interactions
XAF1 2 2.70E-03 2.7 4.90E-04 2.8 6.30E-04 Negative regulator of members of the IAP
(Inhibitor of Apoptosis Protein) family doi:10.1371/journal.pone.0164117.t003
Table 4. Genes consistently repressed by the 3 Borrelia strains with fold-change values0.5.
Annotation B. afzelii (IBS 17) B. garinii (IBS 6) Bb ss (IBS 19) Description/Function Fold change p-value Fold change p-value Fold change p-value Cell division process
CDC20 0,38 3,97E-06 0,35 1,53E-05 0,41 1,19E-04 Activator of the anaphase promoting complex (APC/C)
PBK 0,50 1,22E-05 0,50 6,95E-05 0,49 5,06E-04 PDZ binding kinase: related to mitogen-activated
protein kinase (MAPKK) family
TOP2A 0,46 3,65E-06 0,44 8,88E-06 0,49 3,47E-04 DNA topoisomerase 2-alpha: role in mitosis and
meiosis for proper segregation of daughter chromosomes
KIF20A 0,40 3,97E-05 0,37 3,21E-05 0,41 3,15E-05 Mitotic kinesin required for chromosome passenger
complex (CPC)-mediated cytokinesis
UBE2C 0,37 1,10E-05 0,36 2,35E-05 0,37 9,67E-05 Ubiquitin-conjugating enzyme E2 C: essential factor of
the anaphase promoting complex/cyclosome (APC/C)
CEP55 0,49 1,39E-04 0,47 7,27E-05 0,48 1,72E-03 Centrosomal protein of 55 kDa: role in mitotic exit and
cytokinesis
LOC399942 0,44 3,17E-03 0,48 7,97E-03 0,49 2,20E-02 Predicted: similar to Tubulin alpha-2 chain, major
constituent of microtubules
ASPM 0,46 8,55E-06 0,44 5,35E-05 0,48 6,88E-05 Probable role in mitotic spindle regulation and
coordination of mitotic processes
LOC100132394 0,36 8,45E-04 0,44 8,59E-03 0,36 4,50E-03 Unknown function
Fig 5. QRT-PCR analysis of mRNA levels induced in human dermal fibroblasts by different species of the B.
burgdorferi sl group. For each batch of fibroblasts,–fibroblasts batch #1 to fibroblasts batch #6 (biological replicates)–,
the mRNA levels of IL-8, CXCL1, IL-6 and SOD2 stimulated during 24h at MOI 100:1 by B. burgdorferi ss IBS19 (white bars), B. garinii IBS6 (gray hatched bars) and B. afzelii IBS17 (black bars) were normalized to theβ-actin (A) or RNA polymerase II (B) housekeeping gene levels and expressed as relative changes in gene transcription compared with untreated cells. Each bar shows the mean±SDs of duplicate values (technical replicates).
Fig 6. QRT-PCR analysis of mRNA levels repressed in human dermal fibroblasts by different species of the B. burgdorferi sl group. For each batch of fibroblasts,–fibroblasts batch #1 to fibroblasts batch #6 (biological replicates)–, the mRNA levels of UBE2C, KIF20A, TOP2A, CEP55 and CDC20 stimulated during 24h at MOI 100:1 by B. burgdorferi ss IBS19 (white bars), B. garinii IBS6 (gray hatched bars) and B. afzelii IBS17 (black bars) were normalized to theβ-actin (A) or RNA polymerase II (B) housekeeping gene levels and expressed as relative changes in gene transcription compared with untreated cells. Each bar shows the mean±SDs of duplicate values (technical replicates).
after transmission, when the inflammatory process is culminating. Indeed, although only a small number of Borrelia are present on tick salivary glands and are then inoculated in the dermis during the feeding process [23,24], an intense replication of Borrelia occurs after inoculation in the host skin at the inoculation site, peaking at day 7 after inoculation [25,
26]. Moreover, setting the stimulation conditions at MOI of 100:1 for 24 hours enabled to yield a potent activation of human dermal fibroblasts while allowing the experimental condi-tions to emulate those of our first study assessing the early transcriptional response (at 24 hours) of human dermal fibroblasts stimulated by three strains of B. burgdorferi ss (MOI of 100:1) [19].
Secondly, the microarray experiments were precisely designed and the batches of fibroblasts tested were carefully chosen. A consensual number of 6 different batches of fibroblasts was used in order to reach a sufficient number of biological replicates [27]. Biases were limited by selecting NHDF batches issued from different donors of various sexes and ages, but all batches were issued from areas with similar transcriptional profiles. Indeed, fibroblasts derived from skin at different anatomical sites display distinct transcriptional patterns and this distinction was maintained in vitro when fibroblasts were isolated from the influence of other cell types, suggesting that fibroblasts at different locations in the body should be considered distinct dif-ferentiated cell types [28,29]. Approximately 8% of all genes transcribed in fibroblasts were dif-ferentially expressed in a site-specific manner and this gene expression signature separates fibroblasts from anterior (rostral) and posterior (caudal) sites of the human body divided by the umbilicus [30]. Thus, in order to avoid the risk of missing transcriptional modifications induced by Borrelia, that could be concealed by the heterogeneity of fibroblasts, we chose, in our experiments, fibroblasts issued from a similar transcriptional area (head and chest).
In a previous study, analyzing the interaction between Borrelia and fibroblasts, we tested 3 strains of the same species (B. burgdorferi ss) that were isolated from various clinical environ-ments (strain 1408 isolated from ACA; strain Pbre isolated from EM; strain N40 isolated from a tick). These 3 strains were shown to induce a very similar inflammatory profile in fibroblasts [19]. Therefore, no specific strain-related pathway has been identified that could be linked to the transcriptional responses elicited by clinical manifestations. We thus concluded that
Borre-lia pathotype has little influence on the fibroblasts response. Since the 3 strains used in our
pre-vious study belonged to the same species, this new study was designed to determine whether the fibroblasts transcriptional response could be differentially influenced by the Borrelia spe-cies. The data obtained in this study indicate that the transcriptional response of fibroblasts is largely comparable whichever the Borrelia species used for cell stimulation. Indeed, almost all the transcripts with the highest transcriptional modulation were consistently stimulated by the 3 tested species. Transcripts that were specifically modulated by only one strain showed only minor modifications. Thus, our results do not reveal the existence of a fingerprint of transcrip-tional changes in fibroblasts that could be associated with a particular Borrelia species. How-ever, even if the overall Borrelia-induced transcriptional modulations in fibroblasts were very similar, an interesting finding of our study was that the level of modification induced by the B.
garinii EM-inducing strain was significantly higher than that of B. afzelii and B. burgdorferi ss
EM-inducing strains tested. Clinical observations previously reported B. garinii-induced EM to have shorter incubation and faster evolution than EM caused by B. afzelii [31,32]. EM lesions caused by B. garinii were also significantly larger than EM lesions caused by B. burgdorferi ss [33] or B. afzelii [32]. Thus, our in-vitro observations are in accordance with the clinical fea-tures described by other authors.
Furthermore, the use of a whole genome microarray experiment combined with the analysis of a large number of replicates (3 strains tested with 6 different batches of fibroblasts) allowed an in-depth exploration of the early fibroblasts’ transcriptional response to Borrelia infection.
The main observation was that the 3 Borrelia species induced the stimulation of a large number of pro-inflammatory genes involved in the innate immune response, with high levels of the chemokines/cytokines CCL2, CXCL1, CXCL2, CXCL6, CXCL10, IL-6, IL-8. This result corrob-orates previous studies reporting the induction of CXCL1, IL6 and IL8 in Borrelia-stimulated primary human fibroblasts in vitro [19,34]. Borrelia-induced stimulation of dendritic cells and macrophage chemoattractants was also demonstrated in skin biopsies of murine models (MCP-1, also referred to as CCL2) [26], as well as in human biopsies of EM and ACA lesions (CCL2, CXCL1, CXCL9, CXCL10, and CCL20) [35,36].
We also found that a large number of type I IFN-related genes was intensively stimulated in fibroblasts by Borrelia. Type I IFNs play a key role in linking the innate and adaptive immune responses. The stimulation of the type I IFN heteromeric receptor initiates an intracellular cascade leading to the phosphorylation of STAT proteins (especially STAT1) which in turn translocate to the nucleus and initiate transcription by binding specific sites in the promoters of IFN-stimulated genes [37]. The induction of type I IFN by Borrelia has been previously shown in human peripheral blood mononuclear cells [38]. A critical role of type I IFN has also been demonstrated in the pathogenesis of Borrelia in murine models of Lyme arthritis [39] involving multiple triggering ligands of the bacteria [40]. Fibroblasts were also previously shown to participate in the local propagation of a robust type I IFN response in joint tissues [41]. Since dermal fibroblasts represent the most abundant cells in the site of Borrelia inoculation by the tick biting pieces, the strong Borrelia-induced stimula-tion of type I IFN-related genes observed in our experimental model could indicate that fibroblasts actively participate in the initiation of the inflammatory response to Borrelia in the skin.
Interestingly, the cytosolic pattern recognition receptor pathway of type 1 IFN induction was also evidenced in fibroblasts stimulated by all 3 studied Borrelia strains. This system is triggered by the presence of double-stranded RNA or double-stranded DNA in the cell cytosol following infection by viruses or intracellular bacteria. The cytosolic pattern recognition receptor pathway eventually induces the activation of early phase type 1 IFNs and other pro-teins implicated in the innate immune response [42]. This finding is interesting since Borrelia is classically considered an extracellular pathogen and that only few experimental data cur-rently support that it can be internalised by human dermal fibroblasts [43,44]. However, con-sidering the complex interplay between pattern recognition receptors and the various cross-regulation of the innate immune receptor signaling [45], this finding should be interpreted with caution since the same IFN-responsive genes and IFN-regulatory factors can be stimu-lated by pattern recognition receptors in different cellular locations. In the interaction between Borrelia and fibroblasts, the transcriptional response of fibroblasts do not seem to be related to the Borrelia species tested, since no species-related transcriptional fingerprint could be brought out. Conversely, the fibroblast response appears to be homogeneous whichever the
Borrelia species or pathotype tested and this cell type seems to play a key role in the
inflamma-tory response elicited by Borrelia. The question of the factors involved in the organotropism of Lyme borreliosis manifestations remains unanswered. Factors related to the host are proba-bly more implicated in the pathogenesis of this complex disease and need to be explored in forthcoming studies.
Supporting Information
S1 Fig. Additional titration results of IL-8 secretion by fibroblasts co-incubated with differ-ent species of the B. burgdorferi sl group. Levels of IL-8 secretion by fibroblasts stimulated by
of the 3 Borrelia strains at 24 hours for fibroblasts batch #3 (A-C) and fibroblasts batch #4 (D-F). NEG: unstimulated fibroblasts. NA: not available data. Each bar shows the mean ± SDs of duplicate values.
(TIF)
S2 Fig. Functional analysis of fibroblasts stimulated by Borrelia. (A) Fibroblasts stimulated
by B. burgdorferi ss IBS19. (B) Fibroblasts stimulated by B. garinii IBS6. (C) Fibroblasts stimu-lated by B. afzelii IBS17.
(PDF)
S3 Fig. Analysis of pathways regulated in fibroblasts by Borrelia. (A) Fibroblasts stimulated
by B. burgdorferi ss IBS19. (B) Fibroblasts stimulated by B. garinii IBS6. (C) Fibroblasts stimu-lated by B. afzelii IBS17.
(PDF)
S1 Table. Primers used for the quantitative RT-PCR.
(PDF)
Acknowledgments
We thank Laurence Zilliox and Danièle Napolitano for their technical assistance in Borrelia cultures, and Abiba Doukani for her help in the submission of microarray data to GEO.
Author Contributions
Conceptualization: FS NB BJ. Data curation: MM FS WC NC. Formal analysis: MM FS WC NC. Investigation: MM FS WC AG CB. Methodology: FS WC SN. Project administration: FS NB BJ. Resources: BJ WC NC. Supervision: FS NB BJ. Validation: FS MM. Visualization: FS MM.Writing – original draft: MM FS NB BJ WC NC SN. Writing – review & editing: FS MM NB BJ SJDM.
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